The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

BACKGROUND: Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells. METHOD: A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation. RESULTS: For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iß levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion. CONCLUSIONS: The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

Nano-Pore Structure and Fractal Characteristics of Shale Gas Reservoirs: A Case Study of Longmaxi Formation in Southeastern Chongqing, China.

Pore structure and fractal dimensions can characterize the adsorption, desorption and seepage characteristics of shale gas reservoirs. In this study, pore structure, fractal characteristics and influencing factors were studied of the Longmaxi formation shale gas reservoir in southeastern Chongqing, China. Scanning electron microscopy was used to describe the characteristics of various reservoirs. High pressure mercury intrusion and low temperature liquid N2 and CO2 adsorption experiments were used to obtain pore structure parameters. V-S model, FHH model and Menger sponge model were selected to calculate the micropore, mesopore and macropore fractal dimensions, respectively. The results show that organic matter pores, inter-granular pores, intra-granular pores and micro-fractures are developed within the shale, and the pore morphology is mostly ink pores and parallel plate pores with aperture essentially in the 1-2 nm and 2-50 nm ranges. Moreover, macropores are the most complex in these samples, with mesopores being less complex than macropores, and the micropores being the simplest. D1 (micropore fractal dimension) ranges from 2.31 to 2.50, D2 (mesopore fractal dimension) ranges from 2.74 to 2.83, D3 (macropore fractal dimension) ranges from 2.87 to 2.95, and Dt (comprehensive fractal dimension) ranges from 2.69 to 2.83 of fractal characteristics. D1 and D2 are mainly controlled by TOC content, while D3 and Dt are mainly controlled by brittle and clay mineral content. These results may be helpful for exploration and the development of shale gas in southeastern Chongqing, China.

Adding dexmedetomidine to ropivacaine for lumbar plexus and sciatic nerve block for amputation of lower limb in high-risk patient-a case report.

The ischemia necrosis of limb frequently requires surgery of amputation. Lumbar plexus and sciatic nerve block is an ideal intra-operative anesthetic and post-operative antalgic technique for patients of amputation, especially for high-risk patients who have severe cardio-cerebrovascular diseases. However, the duration of analgesia of peripheral nerve block is hardly sufficient to avoid the postoperative pain and the usage of opioids. In this case, a 79-year-old man, with multiple cerebral infarcts, congestive heart failure, atrial flutter and syncope, was treated with an above knee amputation because of ischemia necrosis of his left lower limb. Dexmedetomidine 1 µg/kg was added to 0.33% ropivacaine for lumbar plexus and sciatic nerve block in this case for intra-operative anesthesia and post-operative analgesia. The sensory function was blocked fully for surgery and the duration of analgesia maintained 26 hours with haemodynamic stability and moderate sedation. The patient did not complain pain and require any supplementary analgesics after surgery. This case showed that adding 1 µg/kg dexmedetomidine to ropivacaine for lumbar plexus and sciatic nerve block may be a feasible and safe technique for high-risk patients for lower limb surgery of amputation.